The objective of this proposal is to investigate how phosphorylation modulates arrestin activity to control the output of rhodopsin thereby fine tuning the visual signaling, using Drosophila eye as a model system. Visual signaling is a G-protein coupled mechanism in which light activates rhodopsin to initiate a cascade of biochemical events leading to depolarization of photoreceptors. It is well established that the activity of rhodopsin is regulated by arrestin that blocks the interaction with the down-stream G-protein. In Drosophila photoreceptors, there are two distinct arrestin homologues, Arr1 and Arr2. Studies support that Arr1 promotes endocytosis of rhodopsin, whereas Arr2 is required for fast deactivation of the visual response by uncoupling activated rhodopsin. Little is known regarding how Arr1 and Arr2 are modulated in vivo. Both Arr1 and Arr2 undergo light-dependent phosphorylation, and Arr2 was shown phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vitro. To investigate how CaMKII modulates Arr2, first we identified CaMKII in the retina by the Ca2+ dependent autophosphorylation. We show that phosphorylation of CaMKII is greatly enhanced by okadaic acid, suggesting that dephosphorylation of CaMKII is regulated by protein phosphatase 2A (PP2A). Significantly, we found that the catalytic subunit of PP2A can be co-immunoprecipitated by anti- CaMKII antibodies. To gain insights into the role of CaMKII in vivo, we demonstrate that suppression of CaMKII in flies leads to enhanced visual response and abnormal light adaptation. These data indicate that CaMKII exerts a negative regulation of the visual response, possibly by catalyzing phosphorylation of Arr2. In contrast, a reduced activity of PP2A in the mts mutants results in reduced visual response without affecting light adaptation, suggesting that PP2A participates in the regulation of Arr2 and/or CaMKII. Based on these findings, we propose that CaMKII and PP2A catalyze the reversible phosphorylation of both Arr1 and Arr2 to modulate the visual response. Moreover, CaMKII and PP2A form a stable protein complex in the retina for a temporal control of the CaMKII activity. To test these hypotheses, we will (1) characterize the functional contribution of Arr2 phosphorylation, (2) investigate the involvement of CaMKII and PP2A in the reversible phosphorylation of Arr1, (3) explore whether PP2A and CaMKII form a stable protein complex in photoreceptors for a dynamic modulation of the CaMKII activity. Our insights into the in vivo regulation of arrestins in Drosophila will broaden our knowledge on the diverse functions that visual arrestins and -arrestins serve in modulating G-protein coupled receptors (GPCRs) involved in diverse signaling pathways. Considering the contribution of GPCRs in many aspects of human physiology and pathophysiology, one may be able to fine-tune the activity of arrestins for improving or optimizing the outputs of GPCRs. In this regard, visual arrestins may present a tangible drug target for future pharmacological intervention to prevent retinal degeneration and to enhance visual acuity. PUBLIC HEALTH RELEVANCE: We would like to investigate regulation of visual arrestins by reversible phosphorylation by a combined genetics, biochemical, and electrophysiological analysis. We propose that CaMKII, by promoting phosphorylation of visual arrestins, is critical for the negative regulation of the visual response. Moreover, the activity of CaMKII may be tightly modulated by PP2A.